Previous studies have shown that the chicken βB1-crystallin promoter (−434/+30) contains all of the signals necessary to specifically direct high level expression of heterologous genes to the lens fiber cells of mice. In the present study, the mouse βB1-crystallin gene was cloned, and its regulation was investigated to further elucidate the mechanisms controlling lens fiber cell-specific gene expression. Phylogenetic footprinting analysis of the 5′ flanking sequence from the mouse, rat, human and chicken βB1-crystallin genes identified several known and putative functional cis elements including the PL2 element which is required for lens-specific expression of the chicken βB1 promoter. Surprisingly, however, all six mouse βB1-crystallin/CAT constructs tested (−1493/+44, −1493/+30, −870/+30, −250/+30, −135/+30 and −98/+30) were inactive in three different mammalian lens-derived cell lines while only the −870/+30 and −98/+30 constructs were active in chicken primary patched lens epithelial cells. In contrast, the chicken βB1-crystallin promoter (−434/+30) was transcriptionally active in all lens-derived cells tested. Transgenic mice harboring a mouse βB1-crystallin −1493/+44 CAT construct did express the transgene specifically in lens fiber cells, however, at lower levels than that previously reported for a chicken −434/+30 CAT construct. These data suggest that, as in other crystallin genes, the regulatory signals controlling lens fiber cell-specific expression are conserved between chicken and mouse. However, the inability of the mouse βB1-crystallin promoter to function in mammalian lens-derived cultured cells implies that this gene has acquired additional cis-regulatory elements to ensure lens fiber cell specificity.